Nucleoside drugs are prodrugs. To be efficacious (or toxic), they must first be transported into the cell by the nucleoside transporters where they are phosphorylated to their active or toxic metabolites. For example, the antiviral drug fialuridine (FIAU), and the anti-HIV dideoxynucleosides (e.g. AZT) must be phosphorylated intracellularly to produce their antiviral efficacy. However, these same triphosphates can also cause toxicity, the most prominent being mitochondrial toxicity. In the case of FIAU, in a Phase II clinical trial, the mitochondrial toxicity of this drug resulted in hepatic failure and death. Amazingly, this toxicity was not predicted from preclinical toxicity studies in mice and rats. Until recently, the mechanism by which these hydrophilic nucleosides (e.g. FIAU) traverse the tight mitochondrial membrane to produce their mitochondrial toxicity was not known. We have obtained some exciting preliminary data that show that the human equilibrative nucleoside transporters (hENTl and HENT2) are expressed in the mitochondrial membrane and transport these nucleoside drugs into the mitochondria from the cytosol. In contrast, their mouse counterparts, mENTl and mENT2, appear NOT to be localized to the mitochondria. Based on these preliminary data, we have hypothesized that the localization of hENTl 12 in the mitochondrial membrane facilitates the entry of nucleosides into the mitochondrial compartment where they are phosphorylated to produce their toxicity. In addition, the difference in mitochondrial localization of human and mouse ENTs explains the inter-species difference in the mitochondrial toxicity of FIAU. Therefore, the long-term goals of this proposal are to determine the role of the equilibrative nucleoside transporters in the in vitro and in vivo mitochondrial toxicity of nucleoside drugs. Hypothesis: Expression of nucleoside transporters in the mitochondrial membrane is a significant determinant of the in vivo mitochondrial toxicity of nucleoside drugs. To test this hypothesis, our specific aims will be: 1) To determine the role of mitochondrial expression of hENT1/2 in the in vitro mitochondrial toxicity of nucleoside drugs 2) To determine the role of mitochondrial expression of hENT1/2 in the in vivo mitochondrial toxicity of FIAU by measuring FIAU hepatotoxicity in wild-type and transgenic mice expressing hENT1 or hENT2 ONLY on the mitochondria.